In the last century there were three major outbreaks of pandemic influenza, and the possibility of future outbreaks remains very high. According to the CDC, the next pandemic will likely result in up to 200 million infections, resulting in 88,000 to 300,000 deaths. In addition to natural transmission, recent years have seen the emergence of bioterrorism as an additional threat. Pandemic influenza is listed as a category C pathogen by the Government's biodefense taskforce. Vaccination represents the most effective mechanism for combating the threat of pandemic influenza; however, current influenza vaccines are not likely to provide adequate protection. We are developing a vaccine candidate based on the highly conserved extracellular domain of the M2 protein, which has the potential to elicit protective immune responses against all influenza A strains. The specific aims of this proposal focus on advancing our understanding of the mechanism and universality of this approach and engineering the vaccine candidate to exhibit increase potency. Specifically, we intend to elucidate the mechanism of anti-M2e antibody associated protection, which we have shown thus far to be antibody mediated, using both in vitro and in vivo techniques. A clearer understanding of the mechanism of protection will be valuable for selecting optimal adjuvant formulations, designing human clinical studies, and establishing correlates of protection for use in human Phase UII studies. While M2 is highly conserved, strains have been identified that contain mutations that could potentially compromise the universality of the vaccine. We intend to investigate the effects of known, naturally occurring M2 mutations on protection using both in vitro and in vivo techniques. Further, we will attempt to improve the potency of the vaccine by incorporating a conserved, broadly HLA-reactive human Th epitope from the nucleoprotein of influenza A. Once a lead candidate vaccine has been identified, we will initiate process development work. This will begin with the manufacture of GMP master and working cell banks, followed by fermentation and purification optimization. This work is expected to yield a manufacturing process that can be transferred to our GMP manufacturing facility to provide high quality protein for formulation, stability, and animal safety toxicology studies.